PubMed · 15055587
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Mark Ptashne. 2004-01-23. Two "what if" experiments.. https://doi.org/10.1016/s0092-8674(03)01076-6
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Terminase enzymes are common to both prokaryotic and eukaryotic double-stranded DNA viruses and are responsible for packaging viral DNA into the confines of an empty procapsid shell. In all known cases, the holoenzymes are heteroligomers composed of a large subunit that possesses the catalytic activities required for genome packaging and a small subunit that is responsible for specific recognition of viral DNA. In bacteriophage lambda, the DNA recognition protein is gpNu1. The gpNu1 subunit interacts with multiple recognition elements within cos, the packaging initiation site in viral DNA, to site-specifically assemble the packaging machinery. Motor assembly is modulated by the Escherichia coli integration host factor protein (IHF), which binds to a consensus sequence also located within cos. On the basis of a variety of biochemical data and the recently solved NMR structure of the DNA binding domain of gpNu1, we proposed a novel DNA binding mode that predicts significant bending of duplex DNA by gpNu1 (de Beer et al. (2002) Mol. Cell 9, 981-991). We further proposed that gpNu1 and IHF cooperatively bind and bend viral DNA to regulate the assembly of the packaging motor. Here, we characterize cooperative gpNu1 and IHF binding to the cos site in lambda DNA using a quantitative electrophoretic mobility shift (EMS) assay. These studies provide direct experimental support for the long presumed cooperative assembly of gpNu1 and IHF at the cos sequence of lambda DNA. Further, circular permutation experiments demonstrate that the viral and host proteins each introduce a strong bend in cos-containing DNA, but not nonspecific DNA substrates. Thus, specific recognition of viral DNA by the packaging apparatus is mediated by both DNA sequence information and by structural alteration of the duplex. The relevance of these results with respect to the assembly of a viral DNA-packaging motor is discussed.
The performances of Bayesian inference to predict the sequence of DNA molecules from fixed-force unzipping experiments are investigated. We show that the probability of misprediction decreases exponentially with the amount of collected data. The decay rate is calculated as a function of biochemical parameters (binding free energies), the sequence content, the applied force, the elastic properties of a DNA single strand, and time resolution.
When mixed together, DNA and polyaminoamide dendrimers form fibers that condense into a compact structure. We use optical tweezers to pull condensed fibers and investigate the decondensation transition by measuring force-extension curves (FECs). A characteristic force plateau (around 10 pN) and hysteresis between the pulling and relaxation cycles are observed for different dendrimer sizes, indicating the existence of a first-order transition between two phases (condensed and extended) of the fiber. Upon salt variation FECs change noticeably confirming that electrostatic forces drive the condensation transition. We propose a simple model for the decondensing transition that qualitatively reproduces the FECs and which is confirmed by atomic force microscopy images.